The most proximal known biochemical consequence of ligation of the T cell antigen receptor (TCR) is activation of several protein tyrosine kinases (PTK). One substrate of the TCR-mediated PTK is phospholipase Cgamma1. Tyrosine phosphorylation of this molecule increases its enzymatic activity and leads to the generation of phosphatidylinositol derived second messengers. More recently it has been shown that TCR ligation also results in activation of ras. It remains unclear how the TCR-activated PTK signaling pathway is coupled to ras. Work on other receptors characterized by PTK activity (such as the insulin, platelet derived growth factor, and epidermal growth factor receptors) has shown that PTKs may couple with ras via several recently characterized molecules including Grb2, a 25kD protein consisting of a src homology 2 (SH2) domain flanked by two src homology 3 (SH3) domains. Interestingly, the molecular details of how the PTK link with ras differs for each receptor. Our laboratory is interested in examining the biochemistry and molecular biology of how the TCR couples to its signal transduction pathways. We have identified four substrates of the TCR-stimulated PTK which bind to Grb2 fusion proteins in vitro. Two of these substrates (pp38 and pp65) appear to associate with Grb2's SH2 domain, while the other two (pp76 and pp116) require Grb2 SH3 domain sequences for binding. Amino acid sequence from purified pp76 and pp116 indicates that both proteins are novel substrates of the TCR-stimulated PTK. The goals of this project are to complete the cloning of cDNA encoding pp76 and pp116 and to characterize these molecules at the DNA and protein level. We propose also to investigate expression of these proteins in various tissues. Additionally, experiments are planned to investigated the physiologic relevance of the association of pp76 and/or pp116 with Grb2 and to determine the molecular basis of the protein-protein interactions. Experiments are also described to investigate other molecules in T cells which may associate with pp76 and pp116. It is hoped that information gleaned from these studies will increase our understanding of how the TCR couples with its signaling machinery. It is also hoped that our work will be applicable to questions of how receptors transduce their signals in general. This information should be helpful in increasing our understanding of how signaling processes may go awry leading to pathoogic conditions.